Anti-fibroblast growth factor-8 monoclonal antibody

ABSTRACT

Described is a monoclonal antibody capable of specifically binding to fibroblast growth factor-8 (FGF-8) which is related to growth factors of hormone-dependent tumor cells such as prostatic cancer and breast cancer, and inhibits the FGF-8 activity. The monoclonal antibody is useful in analyzing the role and biological function of FGF-8 in hormone-dependent tumor cells such as prostatic cancer and breast cancer and can be beneficial in the treatment of these cancers.

This application is a division of Ser. No. 08/832,236 filed Apr. 3,1997, now U.S. Pat. No. 5,952,472.

FIELD OF THE INVENTION

The present invention relates to a monoclonal antibody whichspecifically binds to fibroblast growth factor-8, has a neutralizationactivity, namely it inhibits activity of fibroblast growth factor-8, andtherefore is useful for the morbid state analysis and treatment of humantumor cells grown through induction by fibroblast growth factor-8.

BACKGROUND OF THE INVENTION

Androgen induced growth factor (AIGF) is a factor isolated in 1992 froma culture supernatant of a mouse mammary tumor cell line SC-3 [(ShionogiCarcinoma-3: J. Steroid Biochem., 27, 459 (1987)] which shows sexhormone-dependent growth. AIGF is a growth factor which is induced andproduced by androgen stimulation and activates growth of SC-3 cells inan autocrine manner [Proc. Natl. Acad. Sci., 89, 8928-8932, (1992)]. Theresults of gene cloning efforts revealed that it has a homology of 30 to40% with the FGF family at the amino acid level, and it was namedfibroblast growth factor-8 (hereinafter referred to as “FGF-8”).Thereafter, human FGF-8 was cloned from a human placenta genomic libraryusing mouse FGF-8 as a probe, which coincided with the mouse FGF-8 by afactor of 85% at the nucleotide level and 100% at the amino acid level[FEBS Letters, 363, 226 (1995)]. It has been assumed that sex hormoneinduced growth factors would exhibit an autocrine role in tumors such asprostatic cancer, and breast cancer which show sex hormone-dependentgrowth, and the isolation and cloning of FGF-8, though in a mousesystem, was the first evidence of such a mechanism. It is probable thatFGF-8 also plays a role in carcinogenesis and tumor growth in humans bya similar mechanism, but clear evidence has not yet been obtained.However, since expression of FGF-8 m-RNA can be found in several humantumor cell lines of prostatic cancer and breast cancer and enhancementof cell growth can be observed when FGF-8 expressed in CHO cells isadded to the culture system of these cell lines or a cell line offibroblasts [FEBS Letters, 363, 226 (1995)], it is highly possible thatFGF-8 is one of the growth factors which act on sex hormone-dependenttumors in an autocrine or paracrine manner.

In consequence, an antibody specific for FGF-8 is useful for theanalysis of the role and biological function of FGF-8 in theabove-mentioned tumor cells, and also for the diagnosis of prostaticcancer, breast cancer and the like by immunological detection. Also, itappears that the antibody having a neutralization activity would beuseful in studying biological activities of FGF-8 and effective intreating the cancers.

To date the isolation of a monoclonal antibody specific for FGF-8 hasnot been reported.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a monoclonal antibodywhich specifically binds to FGF-8 and inhibits the activity of FGF-8that has a possibility of being a growth factor of hormone-dependenttumors such as prostatic cancer and breast cancer.

The inventors of the present invention have obtained a monoclonalantibody by preparing hybridomas using a partial peptide of FGF-8 as theimmunogen, establishing a hybridoma strain capable of producing amonoclonal antibody which specifically binds to the peptide, culturingthe hybridoma in a medium or administering it to an animal to induce anascites tumor and then collecting the resulting culture supernatant orascitic fluid. A Western blotting test using the monoclonal antibodyconfirmed that the antibody can bind to FGF-8 protein, when themonoclonal antibody was added to a culture broth of a mouse mammarytumor cell line SC-3 capable of showing sex hormone-dependent growth,and confirmed that the monoclonal antibody can inhibit the FGF-8activity, namely it neutralizes FGF-8.

These and other objects of the present invention have been attained by amonoclonal antibody which specifically binds to FGF-8 and inhibits FGF-8activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the bindability of anti-FGF-8 monoclonal antibody KM 1334for antigens in an enzyme immunoassay (the term “anti-FGF-8 monoclonalantibody” as used herein means monoclonal antibody which specificallybinds to FGF-8). In the drawing, the black quadrangle indicates thebindability for THY-FGF-8 (hereinafter referred to as “THY”) peptideantigen, and the white quadrangle for THY-15639 peptide antigen.

FIG. 2 shows the reactivity of anti-FGF-8 monoclonal antibody KM 1334with purified FGF-8 protein in Western blotting.

FIG. 3 shows the effect of anti-FGF-8 monoclonal antibody KM 1334 ininhibiting the activity of FGF-8. In the drawing, the line of growth at0% indicates a value when neither the growth factor nor antibody wereadded. The dotted line indicates activity inhibition by purified mouseIgG used as a control antibody, and the solid line by anti-FGF-8monoclonal antibody KM 1334.

FIG. 4 shows the effect of anti-FGF-8 monoclonal antibody KM 1334 ininhibiting the activity of bFGF. In the drawing, the line of growth at0% indicates a value when neither the growth factor nor antibody wereadded. The dotted line indicates activity inhibition by purified mouseIgG used as a control antibody, and the solid line by anti-FGF-8monoclonal antibody KM 1334.

FIG. 5 shows the effect of anti-FGF-8 monoclonal antibody KM 1334 ininhibiting the activity of testosterone. In the drawing, the line ofgrowth at 0% indicates a value when neither the growth factor norantibody were added. The dotted line indicates activity inhibition bypurified mouse IgG used as a control antibody, and the solid line byanti-FGF-8 monoclonal antibody KM 1334.

DETAILED DESCRIPTION OF THE INVENTION

Monoclonal antibody KM 1334 produced by a hybridoma cell line KM 1334(FERM BP-5451), a monoclonal antibody of the present invention,specifically binds to FGF-8.

(1) Preparation of Antigen

As the antigen necessary for the preparation of the anti-FGF-8monoclonal antibody, FGF-8 producing cells or a cell fraction thereof,or host cells transformed with a DNA fragment which encodes FGF-8,prepared using known techniques by integrating complete or a partialfragment of cDNA which encodes FGF-8 into procaryotic cells (e.g.,Escherichia coli or the like) or eucaryotic cells (e.g., insect cells,mammal cells or the like) can be used as such or as a protein expressedand purified as a fusion protein, as well as a partial peptide of FGF-8synthesized using a peptide synthesizer.

In order to increase immunogenicity, the peptide to be used as theantigen is linked to a carrier protein using a cross-linking agent.Examples of the carrier protein include keyhole limpet hemocyanin(hereinafter referred to as “KLH”), bovine serum albumin (hereinafterreferred to as “BSA”), cycloglobulin and the like. Examples of thecross-linking agent include glutaraldehyde,N-(m-maleimidobenzoyloxy)-succinimide (hereinafter referred to as “MBS”)and the like.

(2) Immunization of Animal and Preparation of Antibody-producing Cells

Animals immunized are not particularly limited as long as a hybridomacan be prepared. Hereinafter, mice and rats are enumerated as specificexamples in the present invention.

Mice or rats of 3 to 20 weeks of age are immunized with the antigenprepared by the method of (1), and antibody-producing cells arecollected from the spleen, lymph node or peripheral blood of theimmunized animal. The immunization is carried out by administering theantigen together with an appropriate adjuvant (for example, completeFreund's adjuvant or a combination of aluminum hydroxide gel withpertussis vaccine) to the animal subcutaneously, intravenously orintraperitoneally. Following the first administration, the antigen isadministered repeatedly 5 to 10 times at intervals of 1 to 2 weeks.Three to 7 days after each administration, a blood sample is collectedfrom the venous plexus of the fundus of the eye, and the serum derivedfrom the sample blood is tested, for example, by enzyme immunoassay[Enzyme-linked Immunosorbent Assay (ELISA), published by Igaku Shoin,1976] to determine whether it binds to the antigen. A mouse or rat whoseserum shows a sufficient antibody titer against the peptide used forimmunization is submitted for use as a source of antibody-producingcells. For submission to fusion between splenocytes and myeloma cells,the spleen of the immunized mouse or rat is excised 3 to 7 days afterthe final administration of the antigenic substance and splenocytes ofthe spleen are collected. The spleen is cut to pieces in a serum-freebasal medium (hereinafter referred to as “washing medium”) andcentrifuged, and the recovered cells are treated with Tris-ammoniumchloride buffer (pH 7.65) for 1 to 2 minutes to eliminate erythrocytes.The remaining cells are then washed with the washing medium and used assplenocytes for cell fusion.

(3) Preparation of Myeloma Cells

Mouse-derived established cell lines are used as the myeloma cells.Thus, for instance, the 8-azaguanine-resistant mouse (BALB/c-derived)myeloma cell lines P3-X63Ag8-U1 (P3-U1) [Current Topics in Microbiologyand Immunology-1; European Journal of Immunology, 6, 511-519 (1976)],SP2/O-Ag14 (SP-2) [Nature, 276, 269-270 (1978)], P3-X63-Ag8653 (653)[Journal of Immunology, 123, 1548-1550 (1979)] and P3-X63-Ag8 (X63)[Nature, 256, 495-497 (1975)] may be used. These cell lines aresubcultured in an 8-azaguanine medium [prepared by supplementingRPMI-1640 medium with glutamine (1.5 mM), 2-mercaptoethanol (5×10⁻⁵ M),gentamicin (10 μg/ml) and fetal calf serum (FCS) (10%) and furthersupplementing the resulting medium (hereinafter referred to as “normalmedium”) with 8-azaguanine (15 μg/ml)]. Three to four days before cellfusion, subculture is performed in the normal medium to thereby ensure acell number of not less than 2×10⁷ cells on the day of cell fusion.

(4) Cell Fusion

The antibody-producing cells obtained as described in (2) and themyeloma cells obtained as described in (3) are respectively washedthoroughly with the washing medium or PBS (1.83 g of disodium phosphate,0.21 g of monopotassium phosphate and 7.65 g of sodium chloride perliter of distilled water, pH 7.2), and mixed in a proportion of 5 to 10antibody-producing cells per myeloma cell. After recovering of the cellsby centrifugation, the cells are thoroughly loosened, a mixture of 2 gof polyethylene glycol-1,000 (PEG-1,000), 2 ml of the washing medium and0.7 ml of dimethyl sulfoxide is added to the cells with stirring in anamount of 0.2 to 1 ml/10⁸ antibody-producing cells at 37° C., thenseveral 1 to 2 ml portions of the washing medium are added at 1- to2-minute intervals, and the whole amount is made 50 ml by furtherwashing. After centrifugation, the recovered cells are loosened gentlyand then suspended in 100 ml of HAT medium [prepared by supplementingthe normal medium with hypoxanthine (10⁻⁴ M), thymidine (1.5×10⁻⁵ M) andaminopterin (4×10⁻⁷ M)]. This suspension is distributed in 100 μlportions into each well of 96-well culture plates, and incubation iscarried out in a 5% CO₂ incubator at 37° C. for 7 to 14 days. Afterincubation, a portion of the culture supernatant is taken from each welland subjected, for example, to enzyme immunoassay which will bedescribed later in (5) or to FACS (fluorescence-activated cell sorter),thereby selecting an antibody which specifically binds to the FGF-8partial peptide. Thereafter, cloning is repeated twice by the limitingdilution method [using HT medium (HAT medium minus aminopterin) for thefirst cloning and the normal medium for the second]. Cell lines forwhich a high antibody titer is constantly observed are selected asanti-FGF-8 monoclonal antibody-producing hybridoma cell lines.

(5) Selection of Anti-FGF-8 Monoclonal Antibody

Examples of the enzyme immunoassay for measuring antibodies include thesandwich method, an immunoenzymatic technique, a solid phase method inwhich an enzyme-labeled second antibody is used, a method in which animmobilized second antibody is used and a solid phase method in which anenzyme/anti-enzyme antibody system is used [Protein, Nucleic Acid andEnzyme, Supplement No.31, p.23 (1987)]. The following describes themethod in which an enzyme-labeled second antibody is used.

Using a cross-linking agent, the antigenic peptide is linked in advanceto a carrier protein which is different from the one used in theimmunization. A human FGF-8 partial peptide and a peptide having ananimo acid sequence which is different from that of the partial peptideare linked to a carrier protein and used as control peptides. Thepeptide in a concentration of 1 to 50 μg/ml is distributed in 10 to 100μl portions into each well of 96-well EIA plates and allowed to standovernight at 4° C. to effect precoating. After blocking is effected withBSA solution or the like, a hybridoma culture supernatant or a purifiedantibody prepared in accordance with the following procedure (6) is usedas a first antibody and distributed in 50 to 100 μl portions into eachwell of the EIA plates, and the reaction is carried out for 2 hours atroom temperature or overnight at 4° C. After washing with PBS or asolution prepared by supplementing PBS with 0.05% Tween-20 (hereinafterreferred to a s “Tween-PBS”), 1 to 50 μg/ml solution of an anti-mouse oranti-rat immunoglobulin antibody labeled with biotin, an enzyme, achemiluminescent substance, a radioactive compound or the like is usedas a second antibody and distributed in 50 to 100 μl portions into eachwell of the EIA plate, and the reaction is carried out at roomtemperature for 1 to 2 hours. After thorough washing, each reactioncorresponding to respective labeling substance of the second antibody iscarried out, and a well which shows a specific bind to the human FGF-8partial peptide is selected as a source of anti-FGF-8 monoclonalantibody.

(6) Preparation of Monoclonal Antibody

The hybridoma cells obtained in (4) capable of producing anti-FGF-8monoclonal antibody are injected intraperitoneally into 8 to 10-week-oldmice or nude mice treated with pristane [by intraperitonealadministration of 0.5 ml of 2,6,10,14-tetramethylpentadecane (pristane)followed by 3 to 10 days of rearing] at a dose of 2×10⁷ to 5×10⁶ cellsper animal. The hybridoma causes an ascites tumor in 10 to 21 days. Theascitic fluid is collected from the mice, centrifuged (3,000 rpm, 5minutes) to remove solid matter and, after salting out with 40 to 50%saturation ammonium sulfate, subjected to a caprylic acid precipitationmethod or passed through a DEAE-Sepharose column, a protein A column ora gel filtration column. Collected fractions of IgG or IgM are pooled togive a purified monoclonal antibody. The subclass of the antibody can bedetermined using a mouse monoclonal antibody typing kit or a ratmonoclonal antibody typing kit. The amount of protein can be determinedby the Lowry method or calculated based on the optical density at 280nm.

(7) Confirmation of the Specificity of the Monoclonal Antibody (WesternBlotting)

Bindability of the anti-FGF-8 monoclonal antibody selected in (5) withthe FGF-8 protein is examined by Western blotting. A culture broth of amouse breast cancer cell line SC-3 at the time of testosteronestimulation or FGF-8 protein in the culture broth or FGF-8 proteinexpressed in CHO cells is purified, fractionated by SDS-PAGE and thenblotted on a nitrocellulose membrane or PVDF membrane. After blockingwith BSA solution, reaction with a culture supernatant containing theanti-FGF-8 monoclonal antibody or 1 to 10 μg/ml of purified antibody iscarried out at room temperature for 2 hours or overnight at 4° C. Afterwashing with PBS or PBS-Tween, a 1 to 50 μg/ml solution of an anti-mouseor anti-rat immunoglobulin antibody labeled with biotin, an enzyme, achemiluminescent substance, a radioactive compound or the like is usedas a second antibody, and the reaction is carried out at roomtemperature for 1 to 2 hours. After thorough washing, each reactioncorresponding to the respective labeling substance of the secondantibody is carried out to confirm that the anti-FGF-8 monoclonalantibody can bind to the band which coincides with the molecular weightof the FGF-8 protein.

(8) Inhibition of FGF-8 Activity by the Monoclonal Antibody

The ability of the anti-FGF-8 monoclonal antibody selected in (5) toinhibit FGF activity is examined by a growth inhibition assay using amouse breast cancer cell line SC-3 or a cell line derived from humanprostatic cancer or breast cancer as the target cells. According to thismethod, the target cells are cultured in a medium containing FGF-8 (1 to100 ng/ml) or testosterone, which is supplemented in advance with aculture supernatant containing the anti-FGF-8 monoclonal antibody orpurified antibody serially diluted to a final concentration of 0.1 to100 μg/ml. After 24 to 72 hours of culturing, the number of viable cellsis measured using an MTT[3-(4,5-dimethyl-2-thiazonyl)-2,5-diphenyl-2H-tetrazolium bromide]solution or a cell counting kit, thereby confirming inhibition of FGF-8activity by the anti-FGF-8 monoclonal antibody.

EXAMPLES

The present invention will now be illustrated in greater detail by wayof Examples, but it should be understood that the invention is notconstrued as being limited thereto.

Example 1

(1) Preparation of Immunogen

A peptide was synthesized by designing an amino acid sequence (SequenceID NO:1) in which cystein was added to the C-terminal of a partial aminoacid sequence of human FGF-8, the 23 position residue to the 46 positionresidue counting from the N-terminal, in order to effect its bindingwith a carrier protein. The peptide synthesis was carried out using amulti-item simultaneous solid layer system automatic peptide synthesizerPSSM-8 (manufactured by Shimadzu Corp.). In order to improveantigenicity, a conjugate of the synthesized peptide with KLH(manufactured by Calbiochem Co.) was prepared and used as the immunogen.That is, KLH was dissolved in PBS to a concentration of 10 mg/ml, and{fraction (1/10)} volume of 25 mg/ml MBS (Nakalai Tesque) was addeddropwise to the KLH solution, followed by 30 minutes of reaction withstirring. Free MBS was removed by passing the reaction solution througha gel filtration column such as a Sephadex G-25 column (Pharmacia) orthe like which has been equilibrated in advance with PBS, therebyobtaining 2.5 mg of a KLH-MBS conjugate. This was further mixed with 1mg of the peptide dissolved in 0.1 M sodium phosphate buffer (pH 7.0),and the mixture was stirred at room temperature for 3 hours to effectthe reaction. After the reaction, the reaction solution was dialyzedagainst PBS-0.5 M NaCl and used as the immunogen.

(2) Immunization of Animals and Preparation of Antibody-producing Cells

A 100 μg portion of the peptide-KLH conjugate prepared by the methoddescribed in Example 1 (1) was administered together with 2 mg ofaluminum gel and 1×10⁹ cells of pertussis vaccine (manufactured by ChibaSerum Institute) to mice of 5 weeks of age (Balb/c). Starting 2 weeksafter the administration, 100 μg of the peptide-KLH conjugate wasadministered once a week in total of 4 times. A blood sample wascollected from the venous plexus of the fundus of the eye, antibodytiter of the serum derived from the sample blood was examined by anenzyme immunoassay which will be described in Example 1 (3), and thespleen was excised from a mouse showing sufficient antibody titer after3 days of the final immunization. The spleen was cut to pieces in MEMmedium (manufactured by Nissui Pharmaceutical), loosened using a pair offorceps and then subjected to 5 minutes of centrifugation at 1,200 rpm.Thereafter, the supernatant was discarded, the obtained precipitate wastreated with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutesto remove erythrocytes, and then the remaining cells were washed threetimes with MEM medium and used for cell fusion.

(3) Enzyme Immunoassay

A conjugate obtained by cross-linking the human FGF-8 partial peptideshown in Sequence ID NO:1 with thyroglobulin in the following manner wasused as the antigen for the assay. That is, 1 mg of the peptide wasdissolved in 0.1 M ammonium acetate buffer and the solution was adjustedto 1 ml by adding 5 mg of THY which has been dissolved in advance in thesame buffer. To this, while stirring, was added dropwise 540 μl of 0.02M glutaraldehyde, followed by 5 hours of stirring at room temperature toeffect the reaction. After the reaction, the reaction solution wasdialyzed overnight against PBS and used as the antigen. A conjugateobtained in the same manner by cross-linking the peptide shown inSequence ID NO:2 with THY was used as a reference antigen. A 10 μg/mlsolution of the prepared conjugate was distributed in 50 μl portions inwells of a 96-well plate for EIA (manufactured by Greiner), and theplate was allowed to stand overnight at 4° C. to effect coating. Afterwashing, 1% BSA-PBS was distributed in 100 μl portions into wells of theplate which was subsequently subjected to 1 hour of reaction at roomtemperature to block the remaining active groups. The 1% BSA-PBS wasthen discarded, and a hybridoma culture supernatant or an immunizedmouse antiserum was distributed in 50 μl portions into wells of theplate, subsequently carrying out 2 hours of reaction. After washing withTween-PBS, a peroxidase-labeled rabbit anti-mouse immunoglobulin(manufactured by DAKO) was distributed in 50 μl portions into wells ofthe plate to carry out 1 hour of reaction at room temperature, the platewas again washed with Tween-PBS, and then color development was effectedusing an ABTS substrate [2,2-azinobis(3-ethylbenzothiazole-6-sulfonicacid) ammonium] solution and absorbance at OD₄₁₅ was measured (NJ 2001;Japan Intermed).

(4) Preparation of Mouse Myeloma Cells

The 8-azaguanine-resistant mouse myeloma cell line P3-U1 was cultured inthe normal medium and not less than 2×10⁷ cells were thereby secured atthe time of cell fusion and submitted for cell fusion as a parent line.

(5) Preparation of Hybridoma

The mouse splenocytes obtained in Example 1 (2) and the myeloma cellsobtained in Example 1 (4) were mixed in a ratio of 10:1, and the mixturewas centrifuged (1,200 rpm, 5 minutes). The supernatant was discarded,and the precipitated cells were thoroughly loosened. A solution composedof a mixture of 2 g of polyethylene glycol-1,000 (PEG-1,000), 2 ml ofMEM medium and 0.7 ml of dimethyl sulfoxide was added to the cells withstirring at 37° C. in an amount of 0.2 to 1 ml per 10⁸ mousesplenocytes, followed by the addition of several 1 to 2 ml portions ofMEM medium at 1 to 2 minute intervals. Thereafter, the total volume wasmade 50 ml by addition of MEM medium. After centrifugation (900 rpm, 5minutes), the supernatant was discarded, and the cells were gentlyloosened and then gently suspended in 100 ml of HAT medium by repeateddrawing up into and discharging from a graduated pipette. The suspensionwas distributed in 100 μl portions into wells of a 96-well cultureplate, and incubation was performed in a 5% CO₂ incubator at 37° C. for10 to 14 days. Each culture supernatant was examined by the enzymeimmunoassay described in Example 1 (3) to select wells which showedspecific reaction with the FGF-8 peptide. Using the selected wells,cloning was repeated twice using HT medium and the normal medium,respectively, thereby establishing hybridoma cell lines capable ofproducing the anti-FGF-8 monoclonal antibody. The monoclonal antibody KM1334 shown in FIG. 1 is an example of these hybridoma cell lines.Hybridoma KM 1334 was deposited on Mar. 7, 1996, at the NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki,Japan), and has been assigned the designation FERM BP-5451. In thisconnection, its antibody class was IgG1 determined by an enzymeimmunoassay using a subclass typing kit.

(6) Purification of Monoclonal Antibody

The hybridoma cell line obtained in Example 1 (3) was intraperitoneallyadministered to pristane-treated female nude mice (Balb/c) of 8 weeks ofage at a dose of 5 to 20×10⁶ cells per animal. The hybridoma causedascites tumor in 10 to 21 days. The ascitic fluid was collected fromeach ascitic fluid-carrying mouse (1 to 8 ml per animal), centrifuged(3,000 rpm, 5 minutes) to remove solid matter and then purified by thecaprylic acid precipitation method (Antibodies-A Laboratory Manual, ColdSpring Harbor Laboratory, 1988) to obtain purified monoclonal antibody.

Example 2

(1) Examination of the Specificity of the Monoclonal Antibody by WesternBlotting

The FGF-8 protein purified from a culture broth of the mouse breastcancer cell line SC-3 at the time of testosterone stimulation wasfractionated by SDS-PAGE in an amount of 0.1 μg per lane and thenblotted on a PVDF film in the usual way. After blocking with 1% BSA-PBS,reaction with the anti-FGF-8 monoclonal antibody KM 1334 (10 μg/ml) ornormal mouse serum used as a control antibody (×500) was carried out atroom temperature for 2 hours or overnight at 4° C. After thoroughwashing with Tween-PBS, reaction with a peroxidase-labeled anti-mouseimmunoglobulin antibody (manufactured by DAKO) was carried out at roomtemperature for 1 hour. After thorough washing with Tween-PBS, reactionwith ECL (manufactured by Amersham) reagent was carried out for 1minute, excess reagent was removed and then detection was effected bysensitizing the treated film for about 10 seconds to 2 minutes. As shownin FIG. 2, KM 1334 bound to FGF-8. Although FGF-8 has a theoreticalvalue of 22 K dalton, it was observed as a band of around 30 K daltondue to addition of sugar chains and the like.

(2) Examination on the Inhibition of FGF-8 Activity by the MonoclonalAntibody

The ability of the anti-FGF-8 monoclonal antibody to inhibit FGF-8activity was examined by a growth inhibition assay using the mousebreast cancer cell line SC-3 as the target cells. The SC-3 cells werecultured in a medium containing FGF-8 (50 ng/ml), testosterone (10 nM,manufactured by Nakalai Tesque) or basic fibroblast growth factor(hereinafter referred to as “bFGF” (1 ng/ml, manufactured by Pepro TechInc.)). In this case, the medium was supplemented in advance with theanti-FGF-8 monoclonal antibody KM 1334 serially diluted to a finalconcentration of 0.1 to 100 μg/ml. As a control antibody, purified mouseIgG (manufactured by Sigma) was used. After 72 hours of culturing, thenumber of viable cells was measured by an MTT method. That is, a 5 mg/mlsolution of MTT[3-(4,5-dimethyl-2-thiazonyl)-2,5-diphenyl-2H-tetrazolium bromide] inPBS was distributed in 10 μl portions into wells of culture plate andincubated at 37° C. for 4 to 5 hours, 0.04 N HCl-isopropanol wasdistributed in 150 μl portions into the resulting wells and then theplate was shaken at 37° C. for 1 to 2 hours. The formed pigment was thensolubilized and its absorbance at OD₅₄₀ nm was measured. Results ofinhibition against activity of FGF-8, bFGF and testosterone are shown inFIGS. 3, 4 and 5, respectively. As shown in FIG. 3, KM 1334 inhibitedthe growth of SC-3 cells by FGF-8 in a dose-dependent manner, but, asshown in FIG. 4, KM 1334 did not show dose-dependent inhibition of SC-3cells by bFGF. In consequence, it was confirmed that KM 1334 inhibitsthe FGF-8 activity specifically. In addition, as shown in FIG. 5, KM1334 partially inhibited the growth of SC-3 cells by testosterone, butnot completely at an antibody concentration of 100 μg/ml.

The present invention provides a monoclonal antibody which specificallybinds to FGF-8 and inhibits FGF-8 activity. The monoclonal antibody ofthe present invention is useful in analyzing the role and biologicalfunction of FGF-8 in tumor cells and can be used to diagnose prostaticcancer, breast cancer and the like by immunological detection.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

This application is based on application No. Hei 8-81754 filed in Japan,the content of which is incorporated hereinto by reference.

2 1 25 PRT Artificial Sequence Description of Artificial Sequenceartificial sequence is protein 1 Gln Val Thr Val Gln Ser Ser Pro Asn PheThr Gln His Val Arg Glu 1 5 10 15 Gln Ser Leu Val Thr Asp Gln Leu Cys 2025 2 15 PRT Artificial Sequence Description of Artificial Sequenceartificial sequence is protein. 2 Cys Ala Thr Ala Ala Asp Gln Glu LysAsn Pro Glu Gly Asp Gly 1 5 10 15

What is claimed is:
 1. A pharmaceutical composition comprising amonoclonal antibody which specifically binds to SEQ ID NO:1, found inthe N terminal region of human fibroblast growth factor-8, and whichinhibits activity of fibroblast growth factor-8 and a pharmaceuticallyacceptable carrier.
 2. The pharmaceutical composition as claimed inclaim 1, wherein the monoclonal antibody is a monoclonal antibodybelonging to the IgG1 subclass produced by hybridoma FERM BP-5451.
 3. Acomposition comprising a monoclonal antibody which specifically binds toSEQ ID NO:1, found in the N terminal region of human fibroblast growthfactor-8, and which inhibits activity of fibroblast growth factor-8 andan carrier.
 4. The composition as claimed in claim 3, wherein themonoclonal antibody is a monoclonal antibody belonging to the IgG1subclass produced by hybridoma FERM BP-5451.